Fragile X Syndrome (FXS) is the most common heritable single gene cause of autism spectrum disord... more Fragile X Syndrome (FXS) is the most common heritable single gene cause of autism spectrum disorder (ASD). FMR1-KO mice mimic the etiology and phenotypic manifestations of FXS. Neuroanatomical changes in specific brain regions have been reported in clinical settings and in preclinical models. FMR1-KO mice have been generated in different strains including C57Bl/6 (B6) and FVB. Mice on different genetic backgrounds have stable yet distinct behavioral phenotypes that may lead to unique gene-strain interactions on brain structure. Previous magnetic resonance imaging (MRI) studies have examined FMR1 knockout male mice on a B6 and found few differences compared to wild-type mice. Here, we examine brain volumes in FMR1 knockout male mice on a FVB background using high resolution (multi-channel 7.0Tesla) MRI. We observe multiple differences in the neuroanatomy of male FMR1-/y mice on a FVB background. Significantly larger relative volume (% total brain volume) differences were found in major white matter structures throughout the brain. In addition, there were changes in areas associated with fronto-striatal circuitry and other regions. Functional and structural connectivity differences are often seen in human ASD, and therefore, this increased white matter seen in the FMR1-KO-FVB could be highlighting a structural over-connectivity, which could lead to some of the behavioral abnormalities seen with the FMR1-KO-FVB mice. Furthermore, these results highlight the importance of genetic strain contribution to brain structure.
Astrocytes are now recognized as key players in the neurobiology of neurodevelopmental disorders ... more Astrocytes are now recognized as key players in the neurobiology of neurodevelopmental disorders such as Fragile X syndrome. However, the nature of Fragile X astrocyte-mediated control of dendrite development in subtypes of hippocampal neurons is not yet known. We used a co-culture procedure in which wildtype primary hippocampal neurons were cultured with astrocytes from either a wildtype or Fragile X mouse, for either 7, 14 or 21days. The neurons were processed for immunocytochemistry with the dendritic marker MAP2, classified by morphological criteria into one of five neuronal subtypes, and subjected to Sholl analyses. Both linear and semi-log methods of Sholl analyses were applied to the neurons in order to provide an in depth analysis of the dendritic arborizations. We found that Fragile X astrocytes affect the development of dendritic arborization of all subtypes of wildtype hippocampal neurons. Furthermore, we show that hippocampal neurons with spiny stellate neuron morphology exhibit the most pervasive developmental delays, with significant dendritic arbor alterations persisting at 21days in culture. The results further dictate the critical role astrocytes play in governing neuronal morphology including altered dendrite development in Fragile X.
Newborn DBA/1J mouse neopallium was disaggregated and grown in high cell densities in tissue cult... more Newborn DBA/1J mouse neopallium was disaggregated and grown in high cell densities in tissue culture. In culture, the oligodendrocyte cell precursors are recognized as small refractile cells which use astrocyte precursor cells as a substratum. Using metrizamide density gradients, the oligodendrocyte precursor cells were separated from the astroblasts after 7 days in culture and then transplanted into the cerebellums of neonatal mice. The differentiation of the cultured oligodendrocyte precursors was analyzed in the transplants by nuclear morphometry, light and electron microscopy and immunocytochemistry. Analysis of the experiments indicated that the oligodendrocyte precursor cells, initially grown in culture, differentiated and myelinated host neuronal processes after transplantation. Moreover, the ultrastructure of the transplanted oligodendrocytes resembled mature oligodendrocytes in situ.
Cells from the fetal central nervous system (CNS) of rat embryos survive and differentiate when t... more Cells from the fetal central nervous system (CNS) of rat embryos survive and differentiate when transplanted into the peripheral nervous system (PNS) of adult rats. The experiments described here were aimed at investigating selected molecular and ultrastructural features of dissociated CNS cells from the telencephalon of 12-day-old embryos isolated for long periods of time within PNS segments. Neurons and glia of grafts examined 6-12 months after transplantation into the PNS developed several cytoskeletal abnormalities. In neurons, these changes included Hirano bodies within dendrites and a marked perikaryal immunoreactivity to RT97, a monoclonal antibody that normally recognizes in neuronal processes the phosphorylated 200 kDa protein subunit of neurofilaments. Rosenthal fibres were seen within the glial cells. Similar-looking abnormalities have been described in certain human and animal neurodegenerative diseases and in ageing. Although a relationship between the changes in these long-term neural transplants and such diseases is unknown, these observations provide an opportunity for studying their pathogenesis within laboratory conditions.
The reaction of biotinamine with two equivalents of 2-quinoline aldehyde in the presence of Na(OA... more The reaction of biotinamine with two equivalents of 2-quinoline aldehyde in the presence of Na(OAc)3BH in dichloroethane provides N,N-bis(methylquinoline)biotinamine (L1), a molecule displaying a tridentate donor terminus which has proven effective in coordinating to the {M(CO)3}+ core (M = Tc, Re). Reaction of L1 with (NEt4)2[Re(CO)3Br3] yields [Re(CO)3(L1)]Br, a compound with an absorbance at 350 nm and luminescence emission maxima at 425 and 580 nm. The luminescence lifetime of 11.4 mus, which is associated with the 580 nm emission, is sufficiently prolonged to enable time-gating techniques to be used during in vitro imaging studies and to overcome interference from endogenous fluorescence. Exposure of avidin beads to {Re(CO)3(L1)]Br resulted in binding, which was qualitatively imaged using fluorescence microscopy. The 99mTc analogue [99mTc(CO)3(L1)]+1 was prepared by reacting L1 with [99mTc(CO)3(H2O)3]+1 and purified by HPLC. The 99mTc complex is chemically robust and resistant to cysteine and histidine challenges. This study demonstrates that complementary fluorescent and radioactive biotin-derived probes may be readily prepared to allow direct correlation of in vitro and in vivo molecular imaging studies.
Results and problems in cell differentiation, Sep 21, 2011
Astrocytes have been recognized as a class of cells that fill the space between neurons for more ... more Astrocytes have been recognized as a class of cells that fill the space between neurons for more than a century. From their humble beginnings in the literature as merely space filling cells, an ever expanding list of functions in the CNS now exceeds the list of functions performed by neurons. In virtually all developmental and pathological conditions in the brain, astrocytes are involved in some capacity that directly affects neuronal function. Today we recognize that astrocytes are involved in the development and function of synaptic communication. Increasing evidence suggests that abnormal synaptic function may be a prominent contributing factor to the learning disability phenotype. With the discovery of FMRP in astrocytes, coupled with a role of astrocytes in synaptic function, research directed to glial neurobiology has never been more important. This chapter highlights the current knowledge of astrocyte function with a focus on their involvement in Fragile X syndrome.
Fragile X Syndrome (FXS) is the most common heritable single gene cause of autism spectrum disord... more Fragile X Syndrome (FXS) is the most common heritable single gene cause of autism spectrum disorder (ASD). FMR1-KO mice mimic the etiology and phenotypic manifestations of FXS. Neuroanatomical changes in specific brain regions have been reported in clinical settings and in preclinical models. FMR1-KO mice have been generated in different strains including C57Bl/6 (B6) and FVB. Mice on different genetic backgrounds have stable yet distinct behavioral phenotypes that may lead to unique gene-strain interactions on brain structure. Previous magnetic resonance imaging (MRI) studies have examined FMR1 knockout male mice on a B6 and found few differences compared to wild-type mice. Here, we examine brain volumes in FMR1 knockout male mice on a FVB background using high resolution (multi-channel 7.0Tesla) MRI. We observe multiple differences in the neuroanatomy of male FMR1-/y mice on a FVB background. Significantly larger relative volume (% total brain volume) differences were found in major white matter structures throughout the brain. In addition, there were changes in areas associated with fronto-striatal circuitry and other regions. Functional and structural connectivity differences are often seen in human ASD, and therefore, this increased white matter seen in the FMR1-KO-FVB could be highlighting a structural over-connectivity, which could lead to some of the behavioral abnormalities seen with the FMR1-KO-FVB mice. Furthermore, these results highlight the importance of genetic strain contribution to brain structure.
Astrocytes are now recognized as key players in the neurobiology of neurodevelopmental disorders ... more Astrocytes are now recognized as key players in the neurobiology of neurodevelopmental disorders such as Fragile X syndrome. However, the nature of Fragile X astrocyte-mediated control of dendrite development in subtypes of hippocampal neurons is not yet known. We used a co-culture procedure in which wildtype primary hippocampal neurons were cultured with astrocytes from either a wildtype or Fragile X mouse, for either 7, 14 or 21days. The neurons were processed for immunocytochemistry with the dendritic marker MAP2, classified by morphological criteria into one of five neuronal subtypes, and subjected to Sholl analyses. Both linear and semi-log methods of Sholl analyses were applied to the neurons in order to provide an in depth analysis of the dendritic arborizations. We found that Fragile X astrocytes affect the development of dendritic arborization of all subtypes of wildtype hippocampal neurons. Furthermore, we show that hippocampal neurons with spiny stellate neuron morphology exhibit the most pervasive developmental delays, with significant dendritic arbor alterations persisting at 21days in culture. The results further dictate the critical role astrocytes play in governing neuronal morphology including altered dendrite development in Fragile X.
Newborn DBA/1J mouse neopallium was disaggregated and grown in high cell densities in tissue cult... more Newborn DBA/1J mouse neopallium was disaggregated and grown in high cell densities in tissue culture. In culture, the oligodendrocyte cell precursors are recognized as small refractile cells which use astrocyte precursor cells as a substratum. Using metrizamide density gradients, the oligodendrocyte precursor cells were separated from the astroblasts after 7 days in culture and then transplanted into the cerebellums of neonatal mice. The differentiation of the cultured oligodendrocyte precursors was analyzed in the transplants by nuclear morphometry, light and electron microscopy and immunocytochemistry. Analysis of the experiments indicated that the oligodendrocyte precursor cells, initially grown in culture, differentiated and myelinated host neuronal processes after transplantation. Moreover, the ultrastructure of the transplanted oligodendrocytes resembled mature oligodendrocytes in situ.
Cells from the fetal central nervous system (CNS) of rat embryos survive and differentiate when t... more Cells from the fetal central nervous system (CNS) of rat embryos survive and differentiate when transplanted into the peripheral nervous system (PNS) of adult rats. The experiments described here were aimed at investigating selected molecular and ultrastructural features of dissociated CNS cells from the telencephalon of 12-day-old embryos isolated for long periods of time within PNS segments. Neurons and glia of grafts examined 6-12 months after transplantation into the PNS developed several cytoskeletal abnormalities. In neurons, these changes included Hirano bodies within dendrites and a marked perikaryal immunoreactivity to RT97, a monoclonal antibody that normally recognizes in neuronal processes the phosphorylated 200 kDa protein subunit of neurofilaments. Rosenthal fibres were seen within the glial cells. Similar-looking abnormalities have been described in certain human and animal neurodegenerative diseases and in ageing. Although a relationship between the changes in these long-term neural transplants and such diseases is unknown, these observations provide an opportunity for studying their pathogenesis within laboratory conditions.
The reaction of biotinamine with two equivalents of 2-quinoline aldehyde in the presence of Na(OA... more The reaction of biotinamine with two equivalents of 2-quinoline aldehyde in the presence of Na(OAc)3BH in dichloroethane provides N,N-bis(methylquinoline)biotinamine (L1), a molecule displaying a tridentate donor terminus which has proven effective in coordinating to the {M(CO)3}+ core (M = Tc, Re). Reaction of L1 with (NEt4)2[Re(CO)3Br3] yields [Re(CO)3(L1)]Br, a compound with an absorbance at 350 nm and luminescence emission maxima at 425 and 580 nm. The luminescence lifetime of 11.4 mus, which is associated with the 580 nm emission, is sufficiently prolonged to enable time-gating techniques to be used during in vitro imaging studies and to overcome interference from endogenous fluorescence. Exposure of avidin beads to {Re(CO)3(L1)]Br resulted in binding, which was qualitatively imaged using fluorescence microscopy. The 99mTc analogue [99mTc(CO)3(L1)]+1 was prepared by reacting L1 with [99mTc(CO)3(H2O)3]+1 and purified by HPLC. The 99mTc complex is chemically robust and resistant to cysteine and histidine challenges. This study demonstrates that complementary fluorescent and radioactive biotin-derived probes may be readily prepared to allow direct correlation of in vitro and in vivo molecular imaging studies.
Results and problems in cell differentiation, Sep 21, 2011
Astrocytes have been recognized as a class of cells that fill the space between neurons for more ... more Astrocytes have been recognized as a class of cells that fill the space between neurons for more than a century. From their humble beginnings in the literature as merely space filling cells, an ever expanding list of functions in the CNS now exceeds the list of functions performed by neurons. In virtually all developmental and pathological conditions in the brain, astrocytes are involved in some capacity that directly affects neuronal function. Today we recognize that astrocytes are involved in the development and function of synaptic communication. Increasing evidence suggests that abnormal synaptic function may be a prominent contributing factor to the learning disability phenotype. With the discovery of FMRP in astrocytes, coupled with a role of astrocytes in synaptic function, research directed to glial neurobiology has never been more important. This chapter highlights the current knowledge of astrocyte function with a focus on their involvement in Fragile X syndrome.
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